The Lethal and Sublethal Effects of Lambda-Cyhalothrin and Emamectin Benzoate on the Soybean Pest Riptortus pedestris (Fabricius)

Riptortus pedestris (Fabricius, 1775) (Hemiptera: Alydidae) is a major soybean pest in East Asia that can cause soybean staygreen syndrome. To date, no insecticides have been registered for the control of R. pedestris in China, and these insects are primarily controlled in the field through the application of broad-spectrum insecticides including lambda-cyhalothrin (LCT) and emamectin benzoate (EMB). Here, the lethal and sublethal effects of LCT and EMB on R. pedestris were comprehensively evaluated. LCT and EMB were both found to exhibit high levels of toxicity and concentration-dependent repellent effects for R. pedestris. The exposure of third instar nymphs from the F0 generation to LC30 concentrations of LCT and EMB resulted in a significant increase in the duration of nymph development and adult pre-oviposition period (APOP), together with reductions in fifth instar nymph and adult body weight, longevity, oviposition days, fecundity, vitellarium length, lateral oviduct diameter, and vitellogenin (Vg) gene expression as compared to control treatment. Strikingly, these suppressive effects were transmitted to the F1 generation, which similarly experienced the prolongation of preadult development and the preoviposition period (TPOP). Relative to control-treated populations, the F1 generation for these insecticide-treated groups also exhibited significant decreases in population parameter values. Overall, these data offer new insight into the impact that LCT and EMB treatment can have on R. pedestris, providing a valuable foundation for the application of these pesticides in the context of integrated pest management strategies aimed at soybean crop preservation.


Introduction
The rising need for a larger global food supply has accelerated the agricultural intensification process, leading to the widespread application of insecticides [1].Indeed, insecticide use has emerged as a primary approach to pest control even though alternative strategies are available in many cases [2], with some areas using insecticides as the only approach to pest management.In the Thai horticultural system, for example, over half of all farms are entirely dependent on synthetic insecticides [3].The excessive application of insecticides poses a significant threat to environmental safety and public health, in addition to contributing to the development of insecticide-resistant insects [4,5].It is thus vital that insecticides be applied in a rational manner in order to mitigate these risks, with many recent studies having focused on the comprehensive evaluation of different insecticides used in the context of pest management [6,7].
The most common approaches to assessing the impact of a given insecticide on an insect species rely on the calculation of the median lethal dose (LD 50 ) or lethal concentration (LC 50 ) [8].This approach can enable the effective comparison of the bioactivity levels of various insecticides and provides a metric for use when assessing insect resistance [9].In agricultural ecosystems, however, insects are commonly exposed to sublethal insecticide concentrations as a consequence of the degradation and/or misapplication of these agents [9].Sublethal exposure events have the potential to influence the biology, behaviors, and/or physiology of exposed insects, thus impacting overall population growth [5,6,10,11].Intensive research efforts have explored the sublethal effects of insecticides including neonicotinoids, organophates, and pyrethroids [9].These analyses have revealed that sublethal insecticide exposure can adversely impact the body weight, longevity, and fecundity of target species including Cydia pomonella (L., 1758) (Lepidoptera: Tortricidae) [7], Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) [11], and Plutella xylostella (L., 1758) (Lepidoptera: Plutellidae) [12].In other species, however, hormetic effects wherein certain insecticides exhibit stimulatory effects at sublethal doses have been reported.For example, significant increases in Aphis craccivora Koch, 1854, (Hemiptera: Aphididae) longevity and fecundity were reported following exposure to LC 10 and LC 25 concentrations of flupyradifurone [13].
These effects also have the potential to be transgenerationally transmitted to offspring, resulting in long-lived and potentially permanent impacts on population dynamics [7].The rational application of insecticides in the context of an integrated pest management (IPM) strategy is thus strongly dependent on comprehensive analyses of these sublethal effects.
The bean bug, Riptortus pedestris (Fabricius, 1775) (Hemiptera: Alydidae), is a prominent agricultural pest species that is widely distributed throughout China, Japan, Korea, and other Asian countries [14].These pests feed on over 30 species of plants from 13 families and exhibit a preference for leguminous plants, particularly soybeans [15].Both nymphs and adults can cause damage to soybean pods as a result of their piercing and sucking behaviors, resulting in shriveled or empty pods and pronounced reductions in crop quality and yield [15]. R. pedestris-related crop damage is increasingly common in the Huang-Huai-Huai region, and these pests have been confirmed to cause soybean staygreen syndrome, which is characterized by deferred leaf and stem senescence, deformed pods, and seed abortion [16].At present, however, no insecticides have been registered for the control of R. pedestris in China, and these pests are primarily controlled in the field through the application of broad-spectrum insecticides, particularly pyrethroids and avermectins [17].
Lambda-cyhalothrin (LCT) is a highly effective broad-spectrum pyrethroid insecticide that has been employed to control a range of pest species including Apolygus lucorum (Meyer-Dür, 1843) (Hemiptera: Miridae) [18] and Mythimna separata (Walker, 1865) (Lepidoptera: Noctuidae) [19].LCT functions by targeting sodium channels to induce membrane depolarization and inhibit action potentials, resulting in spasms, paralysis, and death [20].The avermectin family insecticide emamectin benzoate (EMB) is also a common agent used to control pests in a range of crop ecosystems [21].EMB serves as an agonist that can stimulate gamma-aminobutyric acid (GABA)-and glutamate (Glucls)-gated chloride channels, resulting in neuromuscular paralysis and death [22].In addition to their direct lethal effects, both LCT and EMB have been reported to cause sublethal and transgenerational effects in species including M. separata [19], Mamestra brassicae (L., 1758) (Lepidoptera: Noctuidae) [23], and Spodoptera littoralis (Boisduval, 1833) (Lepidoptera: Noctuidae) [24].No studies to date, however, have explored the sublethal effects of LCT and EMB on R. pedestris.The irrational application of insecticides may cause the resistance development and/or re-emergence of R. pedestris, thus failing to control this pest.
In order to explore the lethal and sublethal effects of LCT and EMB on R. pedestris and more rationally guide insecticide application in the field, after initial analyses of the toxicity and repellent effects of these two insecticides, their sublethal effects on developmental duration, body weight, morphological parameters, reproductive parameters, and ovarian development were assessed in exposed R. pedestris.The transgenerational effects of these two insecticides were further explored in these pest species.In addition, vitellogenin (Vg), a yolk protein precursor, is mainly synthesized in the fat body, secreted into the hemolymph, then taken up into developing oocytes, and plays a key role in regulating insect reproduction [25].The expression level of Vg is an important parameter for assessing the sublethal effect of insecticides on reproduction [25].For instance, the egg-laying and Vg expression level of Conopomorpha sinensis Bradley, 1986, (Lepidoptera: Gracillariidae) was significantly suppressed after LC 30 EMB exposure; LC 25 triazophos significantly promoted Vg expression level of Sogatella furcifera (Horváth, 1899) (Hemiptera: Delphacidae) [26].Therefore, the expression level of Vg is also assessed in F 0 and F 1 generations of R. pedestris.Together, these results have the potential to provide a foundation for the more effective IPM-based management of R. pedestris to better improve crop integrity.

Bioassays
LCT and EMB toxicity levels were evaluated using a seedling-dip method akin to the previously reported leaf-dip method [5].Briefly, insecticide stocks were prepared in acetone and diluted with water containing 0.1% (v/v) Tween-80 to six different concentrations (LCT: 20, 10, 5, 2.5, 1.25, 0.625 mg/L; EMB: 50, 25, 12.5, 6.25, 3.125, 1.5625 mg/L).Soybean seedlings with two cotyledons were dipped into these solutions for 20 s, allowed to airdry at room temperature, and transferred into plastic containers.Control seedlings were instead dipped in dH 2 O containing 0.1% (v/v) Tween-80.The roots of these seedlings were wrapped in dH 2 O-soaked absorbent cotton, and nymphs were transferred to the plastic container, which was covered with gauze to prevent their escape.Four replicate treatments were established for each concentration, with 20 3rd instar nymphs being used for each replicate.Mortality was evaluated at 72 h post-treatment.Nymphs were considered deceased if they did not exhibit any physical response when probed with a fine paintbrush.

Dual-Choice Behavior Assays
To evaluate R. pedestris preferences for insecticide-treated soybean seedlings relative to untreated seedlings, a dual-choice behavior assay was conducted using a Y-tube olfactometer consisting of a 25 cm long central tube and two 20 cm lateral arms at a 60 • angle.These two branch tubes were connected to two separate flasks as odor sources.One of these flasks contained a soybean seedling treated with the selected dose of LCT or EMB (LC 90 , LC 70 , LC 50 , LC 30 treatment groups), with the other containing a control seedling treated with dH 2 O containing 0.1% (v/v) Tween-80 (control group).A 3rd instar nymph that had been deprived of food for 24 h was released at the base of the central arm in this Y-shaped tube and monitored for 5 min.All nymphs that traveled across 2/3 of the lateral arm and remained there for at least 5 s were considered to have made a 'choice'.Any nymphs that did not make a choice during this period were removed and recorded as having made 'no choice'.Every five tests, the odor sources for the two Y-tube arms were reversed to control for positional bias, and a clean Y-tube was used after every 10 tests.Individual nymphs were tested a single time, and 100 total 3rd instar nymphs were tested using this approach.

Analyses of the Sublethal Effects of Insecticides on the F 0 Generation
The LC 30 concentrations of LCT and EMB were used when testing the sublethal effects of these insecticides on R. pedestris using the same experimental approach as above.Briefly, 3rd instar nymphs were exposed to LC 30 concentrations of LCT or EMB as the treatment group and dH 2 O containing 0.1% (v/v) Tween-80 as a control group.After 72 h, surviving nymphs were fed insecticide-free soybean seedlings with roots wrapped in soaked cotton and dried seeds, which were changed every 3 days.Four replicates (n = 50 nymphs/replicate) were established for each group.Insect development was assessed on a daily basis from 3rd instar to adult emergence.The body weight of 5th instar nymphs and adults was measured using an electronic balance (0.0001 g accuracy, OHAUS Instruments Ltd., Changzhou, China), as were the body length, thorax width, and abdomen width of adults using a VHX-1000 super-high magnification lens zoom 3D microscope (KEYENCE, Osaka, Japan).For body weight and morphology parameters, three replicates (n = 20 individuals/replicate) were used for each group.
Female and male adults that emerged on the same day were randomly paired and transferred into a plastic container in which they were provided with soybean seedlings and dried seeds.Twenty pairs were established as a single replicate, with three replicates per group.The longevity and fecundity of each individual were recorded daily until all adults had died.
The sublethal effects of LC 30 concentrations of LCT and EMB on ovarian development were evaluated by dissecting the abdominal cavities of adult females from the treatment and control groups.Briefly, at 1, 3, 5, 7, and 9 days post-emergence of adults, pins were used to fix adults in a dissecting Petri dish, and the abdominal cavity was then dissected.Tweezers were used to gently remove ovarian samples, which were washed thrice with phosphate buffered saline (PBS, 137 mmol/L NaCl, 2.7 mmol/L KCl, 8mmol/L Na 2 HPO4•12H 2 O, 1.8 mmol/L KH 2 PO4, 10 mmol/L, at pH 7.4) and imaged with a digital microscope (VHX-1000, Keyence, Chennai, India).The vitellarium length and lateral oviduct diameter were measured for each insect.Three replicates (n = 20 individuals/replicate) were used for each age of each group.

Analyses of Sublethal Effects on the Traits of the F 1 Generation
Potential carryover effects of sublethal LCT and EMB concentrations on the F 1 generation were assessed by randomly collecting 100 eggs laid by adults from each group, including LCT treatment, EMB treatment, and control groups.These eggs were then reared on insecticide-free soybean seedlings and dried seeds, with each egg serving as a single replicate.Life table parameters for nymphs and adults, as well as developmental periods, pre-oviposition, oviposition, and fecundity-related data were recorded on a daily basis until the death of the last surviving adult.Measurements were also taken of the body weight of 5th instar nymphs and adults; body length, thorax width, and abdomen width of adults; vitellarium length; and lateral oviduct diameter.For body weight, morphology, and ovarian development parameters, three replicates (n = 20 individuals/replicate) were used for each group.

Life Table Data Analyses
Population age-specific survival rate (l x ), age-specific fecundity (m x ), net reproductive rate (R 0 ), intrinsic rate of increase (r), finite rate of increase (λ), and mean generation time (T) values were calculated with the following equations [27,28]: where x is the interval in days (d), x = 0, 1, 2, 3, . .., m is the number of stages, m = 1, 2, 3, S xj is the survival rate of R. pedestris from egg development to age x and developmental stage j, and f xj is the mean egg production of R. pedestris at age x and stage j.
The age-stage life expectancy (e xj ), defined as the time an individual of age x and stage j is expected to live, was also computed as follows [29]: The age-stage reproductive value (v xj ), which represents the contributions of individuals of age x and stage j to the future population, was computed as follows [30]: where S iy is the probability of an individual of age x and stage j surviving to age i and stage y.S iy is calculated based on the assumption that S iy = 1.Life history data for the F 1 generation were analyzed using the TWOSEX-MSChart program [31] based on the age-stage, two-sex life table theory [28,32].Standard error values for all parameters were estimated through the use of 100,000 bootstrap replicates.Differences among the LCT, EMB, and control groups were determined with paired bootstrap tests at a 5% significance level.

qRT-PCR
To assess the effects of LCT and EMB on Vg expression in R. pedestris, a qRT-PCR analysis was conducted following exposure to the LC 30 concentrations of these insecticides (treatment groups) and dH 2 O containing 0.1% (v/v) Tween-80 (control group).Adults were collected on days 1, 3, 5, 7, and 9 after emergence for these analyses, which were performed as reported previously [33].Briefly, total R. pedestris RNA was extracted with a TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China), and cDNA was prepared with TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reagents (TransGen, Beijing, China) based on provided directions.This cDNA then served as a template for qPCR using primers specific for Vg and elongation factor 1 alpha (EF1α) as reported by Lee et al. [33].Gene expression levels were normalized to EF1α levels and relative expression was assessed via the comparative C T (∆∆Ct) method.Three replicates (n = 20 individuals/replicate) were used for each age of each group.
The above methodology flow is shown in Figure S1.

Statistical Analysis
The log-probit model was used to establish the LC 30 , LC 50 , LC 70 , and LC 90 values for LCT and EMB when used to treat R. pedestris 3rd instar nymphs.Chi-square tests were used to analyze dual-choice behavior assay results, with a presumed 50:50 distribution.Data collected for LCT treatment, EMB treatment, and control groups from the F 0 generation, including developmental duration, the weight, longevity, body length, thorax width, and abdomen width of adults, adult pre-oviposition period (APOP), oviposition days, fecundity, vitellarium length, lateral oviduct diameter, and Vg expression levels were all analyzed via one-way ANOVAs with Tukey's honestly significant difference (HSD) test, and significance was set at p < 0.05.To check the assumptions of normality and homogeneity of parametric analysis, Kolmogorov-Smirnov and Levene's tests were used, respectively.SPSS 20.0 (IBM, Armonk, NY, USA) was used to conduct all statistical analyses.

LCT and EMB Have Toxic Effects on R. pedestris Nymphs
In initial experiments, the respective LC 50 values of LCT and EMB when used to expose R. pedestris 3rd instar nymphs were calculated to be 2.680 mg/L and 7.681 mg/L (Table 1), respectively.Subsequent experiments were performed using LCT and EMB at LC 30 , LC 50 , LC 70 , and LC 90 concentrations, as appropriate.

LCT and EMB Exhibit Repellant Activity
When exposing soybean seedlings to progressively higher concentrations of LCT, an increase in the preference of nymphs for control group was observed such that the difference was significant for the LC 50 vs control (χ 2 = 4.35, df = 1, p = 0.04), LC 70 vs control (χ 2 = 7.91, df = 1, p = 0.005), and LC 90 vs control comparisons (χ 2 = 10.98,df = 1, p = 0.001) (Figure 1a).Similar trends were also evident for EMB, with third instar nymphs exhibiting a greater preference for control soybean seedlings as compared to those exposed to LC 70 (χ 2 = 4.25, df = 1, p = 0.04) and LC 90 (χ 2 = 7.53, df = 1, p = 0.006) concentrations of EMB (Figure 1b).Data collected for LCT treatment, EMB treatment, and control groups from the F0 gen tion, including developmental duration, the weight, longevity, body length, thorax wi and abdomen width of adults, adult pre-oviposition period (APOP), oviposition days cundity, vitellarium length, lateral oviduct diameter, and Vg expression levels were analyzed via one-way ANOVAs with Tukey's honestly significant difference (HSD) and significance was set at p < 0.05.To check the assumptions of normality and homo neity of parametric analysis, Kolmogorov-Smirnov and Levene's tests were used, res tively.SPSS 20.0 (IBM, Armonk, NY, USA) was used to conduct all statistical analyses

LCT and EMB Have Toxic Effects on R. pedestris Nymphs
In initial experiments, the respective LC50 values of LCT and EMB when used to pose R. pedestris 3rd instar nymphs were calculated to be 2.680 mg/L and 7.681 mg/L (T 1), respectively.Subsequent experiments were performed using LCT and EMB at L LC50, LC70, and LC90 concentrations, as appropriate.

LCT and EMB Exhibit Repellant Activity
When exposing soybean seedlings to progressively higher concentrations of LCT increase in the preference of nymphs for control group was observed such that the di ence was significant for the LC50 vs control (χ 2     When third instar nymphs from the F 0 generation were exposed to LC 30 concentrations of LCT and EMB, the developmental durations for third (F 2, 497 = 125.09,p < 0.001), fourth (F 2, 479 = 133.14,p < 0.001), and fifth (F 2, 479 = 47.74,p < 0.001) instar nymphs were significantly prolonged relative to control group.These treatments were also associated with a significant reduction in the longevity of both males (F 2, 177 = 61.58,p < 0.001) and females (F 2, 177 = 81.42,p < 0.001) compared to control (Table 2).LCT and EMB exposure was also associated with significantly longer development for the egg (p < 0.001), second 3.4.Sublethal Effects of LCT and EMB on R. pedestris Reproductive Parameters, Ovarian Development, and Vitellogenin Expression in the F 0 and F 1 Generations Significant differences were observed in the APOP of the F 0 generation among the insecticide-treated and control groups (F 2, 177 = 49.23,p < 0.001), with the highest value in the EMB treatment group (8.67 ± 0.19 days) and the lowest value in the control group (6.37 ± 0.16 days).Significant reductions in adult oviposition days in the F 0 generation were observed relative to control following the exposure of third instar nymphs to LCT and EMB (F 2, 177 = 92.69,p < 0.001), with a corresponding drop in fecundity in these insecticidetreated groups (F 2, 177 = 62.69, p < 0.001) (Table 2).While the APOP of the LCT (p = 0.13) and EMB (p = 0.31) treatment groups in the F 1 generation was not significantly increased, significant prolongation of the total preoviposition period (TPOP) was observed relative to control (LCT, p < 0.001; EMB, p < 0.001).Significant decreases in the oviposition days of the LCT (p = 0.005) and EMB (p = 0.031) treatment groups was also observed in this generation relative to control group.Significant decreases in the fecundity of the insecticide treatment groups in this generation were also observed (LCT, p < 0.001; EMB, p < 0.001) (Table 3).

Sublethal Effects of LCT and EMB on the Body Weight and Morphology of R. pedestris in the F0 and F1 Generations
Relative to the control group (44.29 ± 0.76 mg), the body weight of fifth instar nym in the F0 generation in the insecticide-treated groups (LCT, 36.43 ± 0.81 mg; EMB, 34.0.71 mg) were significantly reduced (F2, 177 = 44.02,p < 0.001) following the exposur In addition, qRT-PCR analysis results revealed extremely low Vg transcript levels in adults on days 1 and 3, followed by pronounced increases in these levels on days 5, 7, and 9. Relative to the control group, Vg expression levels did not differ significantly in insecticide-treated groups on days 1 (F 2, 6 = 1.19, p = 0.37) or 3 (F 2, 6 = 0.46, p = 0.65) in the F 0 generation, although they were significantly lower on days 5 (F 2, 6 = 15.09,p = 0.005), 7 (F 2, 6 = 9.03, p = 0.016), and 9 (F 2, 6 = 32.37,p = 0.001) in these treatment groups (Figure 2e).Similar results were also observed for the F 1 generation.Exposure to both LCT and EMB was associated with significant reductions in Vg expression on days 5 (F 2, 6 = 5.87, p = 0.04) and 9 (F 2, 6 = 7.08, p = 0.03) relative to the control group (Figure 2f).

Sublethal Effects of LCT and EMB on R. pedestris Population Parameters in the F1 Generation
Next, a detailed examination of the transgenerational impact of LCT and EMB exp sure on R. pedestris population parameters in the F1 generation was conducted.Relative the control group, a significant reduction in the net reproductive rate (R0) was observe for both insecticide treatment groups in the F1 generation (LCT, p = 0.044; EMB, p = 0.041

Sublethal Effects of LCT and EMB on R. pedestris Population Parameters in the F 1 Generation
Next, a detailed examination of the transgenerational impact of LCT and EMB exposure on R. pedestris population parameters in the F 1 generation was conducted.Relative to the control group, a significant reduction in the net reproductive rate (R 0 ) was observed for both insecticide treatment groups in the F 1 generation (LCT, p = 0.044; EMB, p = 0.041).Similarly, significant differences in the intrinsic rate of increase (r) (LCT, p = 0.008; EMB, p = 0.012) and finite rate of increase (λ) values (LCT, p = 0.008; EMB, p = 0.012) were observed when comparing the treatment and control groups, although the mean generation time (T) (LCT, p = 0.051; EMB, p = 0.647) was comparable between groups in the F 1 generation (Table 4).Inter-individual variability with respect to developmental rates contributed to a pronounced overlap of stages when assessing the age-stage survival rate (s xj ) values for R. pedestris in the control and insecticide-treated groups.Peak s xj values in the LCT-treated (third instar: 66.67%; female: 36.78%;male: 37.93%) and EMB-treated (third instar: 61.36%; female: 38.64%; male: 37.50%) groups were similar to those for the control group (third instar: 62.64%; female: 40.66%; male: 37.36%) (Figure 5).The age-specific survival rate (l x ), which represents the odds of a newborn surviving to age x without taking stage into consideration, tended to decrease with age but did not differ significantly between the treatment and control groups.The highest age-stage-specific fecundity (f xj ) values in the LCT-and EMB-treated groups were 5.44 and 5.15 eggs/female/day, respectively, and these values were below those for controls (6.57eggs /female/day).The maximum age-specific fecundity (m x ) in the LCT and EMB treatment groups was 2.76 and 2.61 eggs/individual/day, with this value was below that for controls (3.42 eggs/individual/day).The maximum age-specific maternity (l x m x ) value for controls was also higher than that for the LCT and EMB treatment groups.Age-stage reproductive value (v xj ) curves began to rise rapidly when females began laying eggs, peaking at a higher value for control females (66.75 eggs/female/day) as compared to those in the LCT (59.15 eggs/female/day) and EMB (52.71 eggs/female/day) groups (Figure 6).The maximal age-stage life expectancy (e xj ) values from the egg stage to adulthood in the LCT (female, 64.44 days; male, 68.24 days) and EMB (female, 65.41 days; male, 68.97 days) groups were decreased relative to those of the control group (female, 67.49days; male, 71.21 days) (Figure 7).

Discussion
Insecticides remain an essential component of IPM strategies as they can rapidly and effectively mitigate the harm associated with a broad range of pest species [35,36].In agroecosystems, however, these pesticides are subject to asymmetrical application and degradation such that insects are frequently exposed to sublethal concentrations of these chemicals [37].At sublethal concentrations, these compounds can still have profound effects on the biology, physiology, and biochemistry of exposed pests, thereby modulating overall population dynamics [7,12].Efforts to assess the lethal and sublethal effects of different

Discussion
Insecticides remain an essential component of IPM strategies as they can rapidly and effectively mitigate the harm associated with a broad range of pest species [34,35].In agro-ecosystems, however, these pesticides are subject to asymmetrical application and degradation such that insects are frequently exposed to sublethal concentrations of these chemicals [36].At sublethal concentrations, these compounds can still have profound effects on the biology, physiology, and biochemistry of exposed pests, thereby modulating overall population dynamics [7,12].Efforts to assess the lethal and sublethal effects of different insecticides can inform efforts to rationally apply these compounds, facilitating the improvement of IPM strategies so as to minimize the risk of environmental harm [7].Here, both LCT and EMB were found to be highly toxic to R. pedestris individuals, exhibiting concentration-dependent repellant effects.Sublethal LC 30 concentrations of both LCT and EMB had negative impacts on the developmental duration, body weight, body length, and reproductive traits of R. pedestris in the F 0 generation.Strikingly, these sublethal effects were also partially transmitted to the F 1 generation.
Both LCT and EMB have been found to exhibit pronounced toxic effects on a range of insect species including C. pomonella [7], A. lucorum [18], and M. separata [19].In this study, the LC 50 value of LCT following the exposure of third instar R. pedestris nymphs for 72 h was 2.68 mg/L, consistent with its pronounced toxicity.The cypermethrin has similarly been shown to exhibit a high degree of toxicity against this pest species [37].The LC 50 value of EMB (7.68 mg/L) was 1.87-fold higher than that of LCT, potentially owing to the fact that these insecticides exhibit different modes of action.While the lethality of these compounds is important, there is also a pressing need to better understand the behavioral responses of pests to insecticides [38].Here, third instar R. pedestris nymphs exhibited clear avoidance of LCT and EMB, in line with data from Romero et al. [39], who found that both individuals and groups of Cimex lectularius (L., 1758) (Hemiptera: Cimicidae) avoided dwelling on insecticide-treated filter paper.Maharjan and Jung [40] similarly found that R. pedestris adults avoided nine different insecticides, which included γ-cyhalothrin and cyfluthrin.These data highlight the repellant efficacy of these different compounds under laboratory conditions, providing insight that may guide their application in the context of IPM strategies directed against R. pedestris.
In addition to their direct lethal and repellant effects, insecticides have been shown to have a marked impact on insect development even at sublethal concentrations [7,13].
Here, the exposure of R. pedestris third instar nymphs to LCT and EMB resulted in the significant prolongation of the developmental duration in the nymph stage, while adult longevity was shortened notably as compared to controls.Pyrethroids or avermectins have been reported to exhibit similar sublethal effects in various mite and pest species.Moustafa et al. [23], for example, found that EMB exposure resulted in the significant prolongation of M. brassicae larval development, while Zhang et al. [41] determined that exposing Rhopalosiphum padi (L., 1758) (Hemiptera: Aphididae) to beta-cypermethrin at LC 20 and LC 25 doses respectively prolonged first instar nymph development and reduced the longevity of adults.Khan et al. [42] also observed increases in the duration of larval development and a corresponding reduction in male longevity following the exposure of Panonychus citri (McGregor, 1916) (Acari: Tetranychidae) to EMB at an LC 30 concentration.Significant reductions in the body weight of both fifth instar nymphs and adults were also evident in the insecticide treatment groups relative to controls, in line with findings reported previously for insect species including C. pomonella [7], S. littoralis [24], and Paederus fuscipes Curtis, 1840, (Coleoptera: Staphylinidae) [43].Two different factors may explain these results.First, insecticide intake can cause injury to the midgut, resulting in digestive dysfunction and metabolic disorder [21], thereby reducing food consumption and nutrient intake.Secondly, insecticide-exposed nymphs devote more energy to detoxification and various other defense mechanisms such as antioxidant defenses at the expense of growth and development [44,45].Further research will be essential to clarify the intrinsic mechanisms that underlie LCT-and EMB-induced developmental inhibition.
Reproduction-associated parameters are also key metrics for use when assessing the sublethal effects of insecticides when used to treat arthropods [7,12,24].Here, sublethal LCT and EMB concentrations resulted in APOP prolongation and reductions in oviposition days and fecundity in adult R. pedestris relative to the control group.The inhibitory effects of insecticides have similarly been observed in prior reports.Pre-oviposition period delays and decreases in the fecundity of adult Nilaparvata lugens (Stål, 1854) (Hemiptera: Delphacidae) were similarly observed following the exposure of second instar larvae to an LC 30 concentration of EMB [43].EMB LC 5 and LC 15 concentrations also resulted in significant APOP prolongation and a reduction in both S. littoralis oviposition days and fecundity [24].LCT treatment also reduced the mean fecundity and oviposition period of C. pomonella at the LC 30 concentration [7].These results align well with the effects of sublethal LCT and EMB concentrations on R. pedestris population growth observed herein.The vitellin (Vn) precursor vitellogenin (Vg) plays a central role in the regulation of reproductive activity in insects such that measuring Vg expression can offer insight into the dynamic mechanisms governing female fecundity [25,33].Here, the exposure of third instar nymphs to LC 30 concentrations of LCT and EMB resulted in the suppression of Vg mRNA expression in adult R. pedestris.Decreased Vg levels, together with the observed reduction in fecundity in the insecticide treatment groups, may highlight the ability of these insecticides to modulate R. pedestris reproduction in part via regulating Vg expression.This result is consistent with the observed decreases in vitellarium length and lateral oviduct diameter values in the insecticide treatment groups as compared to the control group.Comparable findings have also been reported in C. pomonella [7] and C. sinensis [25].These results are not universal, however, with significant increases in the fecundity and Vg expression levels of female M. separata having been observed following exposure to LCT at an LC 50 concentration [19].LC 25 concentrations of triazophos were also associated with significant increases in fecundity and Vg levels in S. furcifera [26].These discrepant results may be attributable to a range of factors, such as species, insecticide mode of action, and utilized insecticide concentrations [36].
To assess whether the sublethal effects of LCT and EMB can persist into the next generation when used to treat parental R. pedestris, developmental and reproductive parameters were also evaluated for the F 1 generation.Following the exposure of the F 0 generation to LC 30 concentrations of LCT and EMB, a significant prolongation of development in the oviposition days and fecundity of adults relative to controls was observed.This is consistent with prior reports for afidopyropen-treated A. gossypii [11], spinetoram-treated P. xylostella [12], and pyriproxyfen-treated Musca domestica (L., 1758) (Diptera: Muscidae) [46].These transgenerational effects are likely the result of epigenetic processes rather than mutations [2].Exposing insects to sublethal insecticide concentrations has been shown to modulate gene expression through epigenetic processes including histone modifications and DNA methylation [2], with these modifications being transmissible to offspring [47], thus resulting in physiological changes via the process of transgenerational epigenetic inheritance [48].The fecundity of the insecticide-treated group was greater for the F 1 generation relative to the F 0 generation, potentially suggesting that these transgenerational effects may weaken across generations such that they may ultimately disappear.However, further research will be needed to confirm this possibility.
Demographic parameters derived from two-sex life table analyses can offer comprehensive insights into insect population dynamics [49].Here, exposure to LCT and EMB at the LC 30 concentration level resulted in significant decreases in the net reproduction (R 0 ), intrinsic rate of increase (r), and finite rate of increase (λ) values of members of the F 1 generation in the insecticide-treated group relative to the control F 1 population.In line with these data, Zhang et al. [41] previously observed a pronounced reduction in these demographic parameters in the F 1 generation of R. padi after exposure of the parental generation of aphids to beta-cypermethrin at an LC 10 concentration level.Moreover, Ghramh et al. [50] reported significant decreases in demographic parameters of the F 1 generation of M. domestica following parental exposure to LC 10 and LC 30 concentrations of LCT relative to the control group.Members of the F 1 generation in the insecticide-treated groups also exhibited significantly reduced f xj , m x , l x m x , e xj , and v xj values relative to controls.Overall, these data indicate that sublethal LCT and EMB concentrations can suppress R. pedestris population growth even in the offspring generation.

Conclusions
The present results revealed that both LCT and EMB caused significant toxicity and exhibited concentration-dependent repellent effects.Exposing third instar nymphs to these two insecticides resulted in significant increases in the duration of nymph development together with significant reductions in longevity, body weight, oviposition days, fecundity, and Vg expression for adult R. pedestris in the F 0 generation.Exposure to LC 30 concentrations of LCT and EMB also resulted in transgenerational suppressive effects in R. pedestris offspring.These data suggest that LCT and EMB can effectively inhibit R. pedestris population growth such that they may be effective tools for the control of R. pedestris infestations.As field conditions are more complex and variable than laboratory settings, however, additional research will be essential to validate and expand upon these results.Additional transcriptomic and metabolomics analyses will also be vital to clarify the mechanistic basis for the sublethal effects of LCT and EMB on R. pedestris.

3. 3 .
Sublethal Doses of LCT and EMB Impact the Developmental Duration and Longevity of R. pedestris in the F 0 and F 1 Generations

Figure 2 .
Figure 2. The sublethal effects of LCT and EMB on R. pedestris reproduction.Vitellarium lengt the F0 (a) and F1 (b) generations.Lateral oviduct diameters in the F0 (c) and F1 (d) generations.V logenin gene expression in the F0 (e) and F1 (f) generation.Three replicates (n = 20 individuals licate) were used for each age of each group.*** (p < 0.01) indicates a highly significant differe ns (p > 0.05) indicates no significant difference; Tukey's HSD test.Different letters above bars cate significant differences based on Tukey's HSD test (p < 0.05).

Figure 2 .
Figure 2. The sublethal effects of LCT and EMB on R. pedestris reproduction.Vitellarium lengths in the F 0 (a) and F 1 (b) generations.Lateral oviduct diameters in the F 0 (c) and F 1 (d) generations.Vitellogenin gene expression in the F 0 (e) and F 1 (f) generation.Three replicates (n = 20 individuals/replicate) were used for each age of each group.*** (p < 0.01) indicates a highly significant difference, ns (p > 0.05) indicates no significant difference; Tukey's HSD test.Different letters above bars indicate significant differences based on Tukey's HSD test (p < 0.05).

Figure 3 .
Figure 3.The sublethal effects of LCT and EMB on R. pedestris body weight.The body weight of fifth instar nymphs from the F0 generation, (b) adults from the F0 generation, (c) fifth instar nymp from the F1 generation, and (d) adults from the F1 generation.Three replicates (n = 20 individ als/replicate) were used for fifth instar nymphs, female, and male in each group.Blue circles, r regular triangles and green inverted triangles, respectively, represent values in the CK, LCT, a EMB groups.Bars with different letters indicate significant differences (p < 0.05) as determined v Tukey's HSD test.

Figure 3 .
Figure 3.The sublethal effects of LCT and EMB on R. pedestris body weight.The body weight of (a) fifth instar nymphs from the F 0 generation, (b) adults from the F 0 generation, (c) fifth instar nymphs from the

Figure 4 .
Figure 4.The sublethal effects of LCT and EMB on R. pedestris morphological parameters.The bod length of adults in the F0 (a) and F1 (b) generations.The thorax width of adults in the F0 (c) and (d) generations.The abdomen width of adults in the F0 (e) and F1 (f) generations.Three replicates = 20 individuals/replicate) were used for each treatment and control of each sex.Blue circles, re regular triangles, and green inverted triangles, respectively, represent values in the CK, LCT, an EMB groups.Bars with different letters indicate significant differences (p < 0.05) as determined v Tukey's HSD test.

Figure 4 .
Figure 4.The sublethal effects of LCT and EMB on R. pedestris morphological parameters.The body length of adults in the F 0 (a) and F 1 (b) generations.The thorax width of adults in the F 0 (c) and F 1 (d) generations.The abdomen width of adults in the F 0 (e) and F 1 (f) generations.Three replicates (n = 20 individuals/replicate) were used for each treatment and control of each sex.Blue circles, red regular triangles, and green inverted triangles, respectively, represent values in the CK, LCT, and EMB groups.Bars with different letters indicate significant differences (p < 0.05) as determined via Tukey's HSD test.

Figure 5 .
Figure 5.The age-stage specific survival rates (s xj ) of R. pedestris in the F 1 generation.Toxics 2023, 11, x FOR PEER REVIEW 14 of 20

Figure 7 .
Figure 7.The age-stage specific life expectancy (exj) of R. pedestris in the F1 generation.

Figure 7 .
Figure 7.The age-stage specific life expectancy (e xj ) of R. pedestris in the F 1 generation.
a Standard error, b 95% confidence intervals.
a Standard error, b 95% confidence intervals.

Table 4 .
Sublethal effects of LCT and EMB on the population parameters of the R. pedestris F 1 generation.